Project Summary/Abstract Human strongyloidiasis is considered the most neglected of the neglected tropical diseases caused primarily by the intestinal parasitic nematode Strongyloides stercoralis, infecting an estimated 300 million people. Chronic infections with S. stercoralis cause abdominal pain, anorexia, diarrhea constipation, urticaria, and itching and are associated with growth stunting in children. Its larvae can also autoinfect the human host, resulting in many generations of autoinfection. In immunosuppressed patients autoinfection can become unregulated, resulting hyperinfection and in severe intensification of symptoms. Hyperinfections may disseminate to organs and tissues causing severe bleeding, small bowel obstruction and colitis, severe pulmonary disease and sepsis, with fatality rates are as high as 80%. Currently we have only one main drug to treat all parasitic stages of S. stercoralis, ivermectin. However, ivermectin resistance in Strongyloides and other intestinal parasitic nematodes is already present, and in some cases rampant, especially in veterinary medicine. Drugs with new modes of action against this deadly parasite are urgently needed. The soil-bacterium Bacillus thuringiensis (Bt) is the number one biological insecticide agent in the world. The insect-active components are crystal (Cry) proteins that kill insects but that are harmless to vertebrates (no effect >1000 mg/kg). Insecticidal Bt Cry proteins are expressed in transgenic food crops (e.g., >80% of USA corn) and are FDA approved for ingestion. The nematode-active Cry protein, Cry5B (related to insecticidal Cry proteins), cures hookworm and Ascaris intestinal nematode infections in large animals and has been studied extensively. More recently, other parasitic nematode-active Cry proteins have been identified, including Cry14A and Cry21A. These are highly effective in vitro against free-living S. stercoralis adults at low doses, raising the possibility that they may represent a new cure for this parasite. This proposal aims to take advantage of 22 known natural amino acid variants of both proteins to find the optimal Cry14A and Cry21A sequences that target this parasite, screening against both free-living adult and parasitic female stages. Once identified, the best Cry14A and Cry21A protein variants will be tested for their ability to cure S. stercoralis infections in vivo in gerbils. The interaction of these proteins with ivermectin-resistant nematodes and, conversely, the interaction of ivermectin with Cry14A resistant nematodes will be investigated. Future plans include dose ranging studies, optimum formulation, detailed engineering and sequence optimization for efficacy and protein yield, studies of mechanism of action and resistance, testing in higher models including natural S. stercoralis infections in non-human primates, and combinatorial studies with ivermectin and other Cry proteins. At the end, we expect to have identified the first new lead anthelmintic against S. stercoralis approval of ivermectin in 1988.